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Phosphoribosylpyrophosphate (PRPP)-less mutants of Escherichia coli
1Enzyme Division, University Institute of Biological Chemistry B, Copenhagen, Denmark.
Abstract:
A DNA fragment encoding kanamycin resistance was inserted in vitro into a plasmid-borne prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli. The resulting plasmids were subsequently transferred to the chromosome by homologous recombination and the haploid strains prs-3::KanR and prs-4::KanR were obtained. These strains were fully viable, but required guanosine, uridine, histidine, tryptophan and nicotinamide mononucleotide. There was no phosphoribosylpyrophosphate synthetase activity or phosphoribosylpyrophosphate pool in the mutant strains. These results show that phosphoribosylpyrophosphate synthetase is dispensable for E. coli.
Insights
Phosphoribosylpyrophosphate synthetase (PRS) is not essential for Escherichia coli viability. Mutant strains lacking PRS activity were viable but required specific nutrient supplements, indicating PRS is dispensable.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Phosphoribosylpyrophosphate synthetase (PRS) is a key enzyme in nucleotide biosynthesis.
- The essentiality of PRS in Escherichia coli has not been definitively established.
Purpose of the Study:
- To investigate the dispensability of phosphoribosylpyrophosphate synthetase (PRS) in Escherichia coli.
- To characterize the metabolic requirements of E. coli strains lacking PRS activity.
Main Methods:
- In vitro insertion of a kanamycin resistance gene into the plasmid-borne prs gene.
- Homologous recombination to transfer the modified prs gene to the E. coli chromosome.
- Phenotypic analysis of resulting haploid mutant strains (prs-3::KanR and prs-4::KanR).
Main Results:
- Haploid E. coli strains lacking detectable PRS activity and intracellular PRS pool were successfully generated.
- These mutant strains exhibited auxotrophy for guanosine, uridine, histidine, tryptophan, and nicotinamide mononucleotide.
- Despite auxotrophy, the mutant strains demonstrated full viability under supplemented conditions.
Conclusions:
- Phosphoribosylpyrophosphate synthetase (PRS) is dispensable for the viability of Escherichia coli.
- The study elucidates the essentiality of PRS in purine and pyrimidine biosynthesis pathways.
- E. coli can survive without PRS activity by utilizing exogenous precursors for nucleotide synthesis.